The present invention relates to a novel hapten-polymer carrier complex comprising a linking unit located so as to not disrupt the antigenic determinant groups of the hapten. The invention further relates to a specific immunoassay for the identification and quantification of pesticides and their degradation products using said hapten.
Pesticides are widely used in many countries. Dichlorbenil (Casoron G(copyright), dichlorobenzonitrile) is a pesticide used as a broad-spectrum herbicide both in agriculture and in urban areas. This herbicide is degraded, mainly to 2,6-dichlorobenzamide (BAM) by biotic or abiotic processes in the hydrosoil (FIG. 1A) (Beynon and Wright, 1972; Montgomery et al., 1972; Verloop, 1972). Chlorthiamid (Prefix(copyright)), a commercial pesticide, also degrades to BAM (FIG. 1D). BAM is very persistent, and is now contaminating a large number of ground-water areas in Europe and the U.S.A.(Brxc3xcsch, 1998). To prevent further leaching of BAM to the drinking water, dichlorbenil is now banned in many countries.
Most BAM-analyses are based on high-performance liquid chromatography (HPLC) and gas chromatography (GC) (van Rossum et al., 1978; Connick and Bradow, 1984) which have detection limits of approximately 0.01 xcexcg/L. Such analyses are very costly for national groundwater-monitoring programs. To lower expenses on pesticide analyses, immunoassays have been developed for several pesticides (Kaufman and Clower, 1995; Issert et al., 1997), but not yet for BAM or the degradation products of dichlorbenil.
The advantage of immunoassays is their extremely high sensitivity, which enables the quantification of pesticides or their degradation products in concentrations lower than 1 xcexcg/L without the need for concentration of samples. Analyses based on HPLC and GC (and GC mass spectroscopy, GC-MS) often require concentration of the sample by evaporation of approximately 2 L of aqueous sample to volumes one thousandth of the original volume.
An immunoassay involves the detection or measurement of an analyte using the specific interaction between an antibody and an antigen. The immune complex that occurs specifically between the antigen and the antibody forms the foundation of such assays. Usually, the antibody is labelled with a tag for detection or quantification purposes. An assay comprising of an enzyme-linked antibody (or antigen) complexing to an immobolised antigen (or antibody) is commonly referred to as an ELISA (enzyme-linked immunosorbent assay).
An immunological process for the assaying a class of herbicides have previously been described (EP 0 300 381 B1). However, this class of compounds does not foresee the use of an appropriate linking unit moiety, nor does it anticipate the class of compounds or haptens described herein. It describes an immunological process for the assay of tri-substituted aromatic rings in which two of the three substituents are fixed 1,3 with respect to one another (meta to one another) whereas the third substituent can be in positions 2, 4, 5, or 6 (ortho or para to either of the other two substituents). Moreover, any of the three substituents can serve to couple to a solid matrix. The hapten, which is the same as the hapten immobilised to the matrix, was used to raise antibodies A, B, or C for the purpose of the assay and that at least two of the said antibodies were reacted separately with a sample supposedly containing said hapten.
Hammock et al (J. Agric. Food Chem. 1984, 32, 1294) investigated the sensitivity and specificity of immunoassays for benzoylphenylureas in relation to heterologous site systems and homologous site systems whereby the coupling site of the hapten was varied or remained constant, respectively.
In U.S. Pat. No. 5,654,178, an antibody useful in an immunoassay for tetrachloroisophthalonitrile is raised. The hapten is coupled to protein via a functional group interchange of a chloro group present on the ring.
A first aspect of the invention relates to a hapten-polymer carrier complex comprising a polymer carrier and a compound of the general formula I 
wherein R2 serves as a linking unit to the polymeric carrier to form the hapten-polymer carrier complex; and wherein R1 is selected from the group consisting of xe2x80x94C(xe2x95x90O)xe2x80x94N(R5)(R5, xe2x80x94N(R5)xe2x80x94C(xe2x95x90O)xe2x80x94OR5, xe2x80x94C(xe2x95x90O)xe2x80x94OR5, xe2x80x94N(R5)xe2x80x94C(xe2x95x90O)(R5, xe2x80x94N(R5)xe2x80x94C(xe2x95x90S)(R5, xe2x80x94C(xe2x95x90S)xe2x80x94n(R5)(R5, xe2x80x94N(R5)xe2x80x94C(xe2x95x90S)xe2x80x94N(R5)(R5, xe2x80x94N(R5)xe2x80x94C(xe2x95x90O)xe2x80x94N(R5)(R5, xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94R5, xe2x80x94C(xe2x95x90S)xe2x80x94OR5, xe2x80x94oxe2x80x94C(xe2x95x90S)xe2x80x94R5, xe2x80x94Oxe2x80x94R5, xe2x80x94N(R5)(R5, xe2x80x94Sxe2x80x94R5, xe2x80x94CH2xe2x80x94R5, xe2x80x94NC, and halogen; where R5 is independently selected from the group consisting of hydrogen, optionally substituted C1-4 alkyl, optionally substituted C1-6 alkenyl, optionally substituted C2-6 alkynyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted C3-C7 cycloalkyl; and wherein X1 and X2 are independently selected from halogens.
In a second aspect, the invention relates to a use of said complex for the identification or quantification of a pesticide and/or its degradation product or products by an immunoassay involving antibodies raised from said hapten-polymer carrier complex.
A second aspect of the invention relates to a method, for the identification or quantification of a pesticide and/or its degradation product or products by an immunoassay involving antibodies raised from a hapten-polymer carrier complex of general formula I, 
wherein X1,X2, R1, R2 are as defined as above; and
a compound of the general formula II immobilised to a solid support 
wherein Z1 is Cxe2x80x94R6, Z2 is Cxe2x80x94R7, Z3 is Cxe2x80x94R8 and Z4 is Cxe2x80x94R8, where at least one of R6-R10 is present and serves as a linking unit for immobilisation of the hapten to the solid support, and the ones of R6-R10 which are present and which do not serve as a linking unit are independently selected from the group consisting of xe2x80x94C(xe2x95x90O)xe2x80x94NH2, xe2x80x94N(R5)(R5), xe2x80x94CN, N(R5)xe2x80x94C(xe2x95x90O)(R5), xe2x80x94N(R5)xe2x80x94C(xe2x95x90S)(R5), xe2x80x94C(xe2x95x90S)xe2x80x94N(R5)(R5, xe2x80x94N(R5)xe2x80x94C(xe2x95x90S)xe2x80x94N(R5)(R5), xe2x80x94N(R5)xe2x80x94C(xe2x95x90O)xe2x80x94N(R5)(R5), xe2x80x94N(R5)xe2x80x94C(xe2x95x90O)xe2x80x94OR5, xe2x80x94C(xe2x95x90O)N(R5)(R5), xe2x80x94C(xe2x95x90O)xe2x80x94OR5, xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94R5, xe2x80x94C(xe2x95x90S)xe2x80x94OR5, xe2x80x94Oxe2x80x94C(xe2x95x90S)xe2x80x94R5, hydrogen, and halogen; and wherein those of R6-R10 which serve as a linking unit are selected from the group consisting of xe2x80x94C(xe2x95x90O)xe2x80x94N(R5)xe2x80x94(CH2)nxe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4, xe2x80x94N(R5)xe2x80x94C(xe2x95x90O)xe2x80x94CH2)nxe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4, xe2x80x94Oxe2x80x94(CH2)nxe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4, xe2x80x94N(R5)xe2x80x94(CH2)nxe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4, xe2x80x94Sxe2x80x94(CH2)nxe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4, xe2x80x94(CH2)n+1xe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4, xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94(CH2)nxe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4, and xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94(CH2)nxe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4; wherein n and m are independent and are integers from 0 to 8; R3 is selected from the group consisting of xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94C(xe2x95x90S)xe2x80x94, xe2x80x94O(xe2x80x94S)xe2x80x94Oxe2x80x94, xe2x80x94N(R5)xe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94C(xe2x95x90O)xe2x80x94N(R5)xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94N(R5)xe2x80x94, optionally substituted C1-4 alkylene, optionally substituted arylene, optionally substituted heteroarylene, an optionally substituted C3-C7 cycloalkylene, R4 is selected from the group consisting of activated forms of a carboxyl, carboxylic acid, amine, acyl halides, activated esters, thiols, disulphides (xe2x80x94Sxe2x80x94Sxe2x80x94R), halides, sulphonates, quinones and imides; and R5 is independently selected from the group consisting of hydrogen, optionally substituted C1-4 alkyl, optionally substituted C2-5 alkenyl, optionally substituted C2-6 alkynyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted C3-C7 cycloalkyl.
A third aspect of the invention relates to kit for an immunoassay comprising a solid support and hapten-polymeric carrier complex as defined above immobilised to said solid support.
The present investigators have surprisingly found that in immunoassays for certain 1,3-dihalo aromatic pesticides, such as dichlorbenil, chlorthiamid and their derivatives and degradation products, linking of said hapten to a polymeric carrier via the 4-position provides for assays with excellent specificity and little cross-reactivity over hapten polymeric carrier complexes whereby the linking moiety is via the functional group at the 2-position or via either of the halo groups native to the pesticide. Furthermore, the location of the linker moiety at the non-activated 4 position provides improved specificity in immunoassays over hapten-polymeric carriers whereby the coupling of the hapten to a polymeric carrier is through an alternative non-activated position.
Specifically, positioning the linker moiety meta to the functional group at the 2 position of a 1,2,3-trisubstituted aromatic hapten, such as 2,6-dichlorbenzamide, for coupling to a polymeric carrier to form a hapten-polymeric carrier complex provides an excellent antigen for raising antibodies which are useful in immunoassays against dichlorbenil and chlorthiamid and their degradation products and derivatives.
The hapten-polymeric carrier complex allows for an immunoassay involving the use of antibodies raised from these novel complexes which are antigenic in animal systems. The raising of antibodies is performed prior to the immunoassay by any number of methods known by persons skilled in the art.
In the present context, the term xe2x80x9chaptenxe2x80x9d is intended to mean a compound which can combine with an antibody, but cannot initiate an immune response unless it is bound to a polymer carrier forming a hapten-polymer carrier complex, said complex serving as an antigen that elicits a specific immune response when introduced into the tissues of an animal. The term xe2x80x9chaptenxe2x80x9d is also intended to mean a compound that upon linking to a polymer carrier to for a hapten-polymer carrier complex, said complex serves as an antigen to an antibody in an immunoassay.
In the present context the term xe2x80x9cC1-4-alkylenexe2x80x9d used alone or as part of another group designates a linear, branched or saturated diradical hydrocarbon group having from one to four carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl.
In the present context the term xe2x80x9cC1-4-alkylxe2x80x9d used alone or as part of another group designates a linear, branched or saturated monoradical hydrocarbon group having from one to four carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl.
In the present context the term xe2x80x9cC2-5-alkylenexe2x80x9d is intended to mean a linear, branched or diradical having from two to five carbon atoms and containing one or more double bonds. Examples of C2-8-alkenyl groups include olefins such as allyl, homo-allyl, vinyl, crotyl. butenyl, pentenyl. Examples of C2-5-alkenyl groups with more than one double bond include butadienyl, pentadienyl as well as branched forms of these. Preferred examples of C2-8-aklenyls are vinyl, allyl and butenyl.
In the present context the term xe2x80x9cC2-6-alkynylxe2x80x9d is intended to mean a linear, branched or monoradical having from two to five carbon atoms and containing one or more double bonds. Examples of C2-6-alkenyl groups include olefins such as allyl, homo-allyl, vinyl, crotyl, butenyl, pentenyl. Examples of C2-6-alkenyl groups with more than one double bond include butadienyl, pentadienyl as well as branched forms of these. Preferred examples of C2-8-alkenyl are vinyl, allyl and butenyl.
In the present context the term xe2x80x9cC2-8-alkynylenexe2x80x9d is intended to mean linear, branched diradical containing from two to five carbon atoms and containing one or more triple bonds. Examples of C2-8-alkynyl groups include acetylene, propynyl, butynyl, pentynyl, hexynyl as well as branched forms of these.
In the present context the term xe2x80x9cC2-8-alkynylxe2x80x9d is intended to mean linear, branched monoradical containing from two to five carbon atoms and containing one or more triple bonds. Examples of C2-8-alkynyl groups include acetylene, propynyl, butynyl, pentynyl, hexynyl as well as branched forms of these.
In the present context the term xe2x80x9carylenexe2x80x9d used alone or as part of another group is intended to mean a biradical aromatic carbocyclic ring or ring system, such as phenyl, naphthyl, anthracyl, phenanthracyl, pyrenyl, benzopyrenyl, fluorenyl, and xanthenyl, preferably phenyl.
In the present context the term xe2x80x9carylxe2x80x9d used alone or as part of another group is intended to mean a monoradical aromatic carbocyclic ring or ring system, such as phenyl, naphthyl, anthracyl, phenanthracyl, pyrenyl, benzopyrenyl, fluorenyl, and xanthenyl, preferably phenyl.
The term xe2x80x9cheteroarylenexe2x80x9d is intended to mean an arylene group where one or more carbon atoms have been replaced with heteroatoms such as nitrogen, sulphur, and/or oxygen atoms. Examples of such heteroaryl groups are oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl, piperidinyl, coumaryl, furyl, quinolyl, indolyl, benzopyrazolyl, and phenoxazonyl.
The term xe2x80x9cheteroarylxe2x80x9d is intended to mean an aryl group where one or more carbon atoms have been replaced with heteroatoms such as nitrogen, sulphur, and/or oxygen atoms. Examples of such heteroaryl groups are oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl, piperidinyl, coumaryl, furyl, quinolyl, indolyl, benzopyrazolyl, and phenoxazonyl.
In the present context the term xe2x80x9cC3-C7 cycloalkylenexe2x80x9d is intended to cover biradical three-, four-, five-, six- and seven-membered rings comprising carbon atoms only (carbocyclic) or carbon atoms together with from 1 to 3 heteroatoms (heterocyclic), wherein the heteroatoms are independently selected from oxygen, sulphur, and nitrogen. Such rings may contain no unsaturated bonds or may contain one or more unsaturated bonds, however, if present, situated in such a way that an aromatic xcfx80-electron system does not arise.
In the present context the term xe2x80x9cC3-C7 cycloalkylxe2x80x9d is intended to cover monoradical three-, four-, five-, six- and seven-membered rings comprising carbon atoms only (carbocyclic) or carbon atoms together with from 1 to 3 heteroatoms (heterocyclic), wherein the heteroatoms are independently selected from oxygen, sulphur, and nitrogen. Such rings may contain no unsaturated bonds or may contain one or more unsaturated bonds, however, if present, situated in such a way that an aromatic xcfx80-electron system does not arise.
The term xe2x80x9chalogenxe2x80x9d includes fluorine, chlorine, bromine and iodine.
In the present context, i.e. in connection with the terms xe2x80x9carylenexe2x80x9d and xe2x80x9cheteroarylenexe2x80x9d, the term xe2x80x9coptionally substitutedxe2x80x9d is intended to mean that the group in question may be substituted one or several times, such as 1 to 5 times, preferably 1 to 3 times, with one or more groups selected from C1-4-alkyl, C1-4-alkoxy, oxo (which may be represented in the tautomeric enol form), carboxy, amino, hydroxy (which when present in an enol system may be represented in the tautomeric keto form), nitro, sulphono, sulphanyl, C1-4-carboxy, C1-4-alkoxycarbonyl, C1-4-alkylcarbonyl, formyl, aryl, arloxy, arloxycarbonyl, arylcarbonyl, heteroaryl, amino, mono- and di(C1-4-alkyl)amino; carbamoyl, mono- and di(C1-4-alkyl)-aminocarbonyl, amino-C1-4-alkyl-aminocarbonyl, mono- and di(C1-4-alkyl)amino-C1-4-alkyl-aminocarbonyl, C1-4-alkylcarbonylamino, cyano, guanidino, carbamido, C1-4-alkanoyloxy, sulphono, C1-4-alkylsulphonyloxy, nitro, sulphanyl, dihalogen-C1-4-alkyl, trihalogen-C1-4-alkyl, halogen, where aryl and heteroaryl representing substituents may be substituted 1-3 times with C1-4-alkyl, C1-4-alkoxy, nitro, cyano, hydroxy, amino or halogen.
In the present context, the term xe2x80x9coptionally substitutedxe2x80x9d in connection with xe2x80x9cC1-4-alkenylenexe2x80x9d, xe2x80x9cC2-5-alkenylenexe2x80x9d, xe2x80x9cC2-4-alkynylenexe2x80x9d, xe2x80x9cC3-C7 cycloalkylenexe2x80x9d, xe2x80x9calkylxe2x80x9d, xe2x80x9cC1-4-alkylxe2x80x9d, xe2x80x9cC2-5-alkenylxe2x80x9d, and xe2x80x9cC2-8-alkynyl is intended to mean that the group in question may be substituted one or more times with hydroxy; C1-4-alkoxy optionally substituted one or more times with halogen, hydroxy, amino, mono- or di(C1-4-alkyl)amino, cyano, and carboxy; halogen; nitro; nitroso; cyano; carboxy; thiolo; C1-4-alkylthio; aryloxyl, heteroaryloxy, arylthio; C1-4-alkylsulphonyl; arylsulphonyl; sulphono (SO3H); sulphino (SO2H); halosulphonyl; isocyano; isothiocyano; and thiocyano.
I. The Compound Used for Immunisation
a) The Linking Unit Moiety
i) The coupling process
As will be understood from examples herein, the position, length and nature of the linking unit of the haptens are of utmost importance for an immunoassay selective for degradation products and derivatives of dichlorbenil and chlorthiamid. The linking unit, optionally upon activation, serves to conjugate a polymer carrier to a hapten for the purpose of the immunisation of the animals. The linking unit may serve to orient the hapten for recognition by the antibody. The linking unit, R2, in formula I, must be of appropriate length and flexibility to orient the hapten relative to the polymer carrier appropriately.
The linking unit, R2, can comprise, at its terminus, of a functional group suitable for coupling to a polymer carrier or of a group suitable for activation. The terminus of the linking unit is either suitable for activation or is an activated species itself (termed a xe2x80x9cpre-activated systemxe2x80x9d) suitable for forming a covalent bond to a polymer carrier.
The person skilled in the art will be aware of a whole array of functional groups responsive to stimuli in photochemical, electrochemical, and chemical processes. Examples of functional groups considered to be photo-, thermo, electrochemical- or chemoreactive suitable for forming a covalent bond to the polymer carrier include, to name but a few, carboxylates, carboxylic acids, acyl halides, activated esters, thiols, disulphides (xe2x80x94Sxe2x80x94Sxe2x80x94R), amines, halides, sulphonates, quinones, diazotized amines, and imides.
In one embodiment of the invention, R2 is selected from the group consisting of xe2x80x94C(xe2x95x90O)xe2x80x94N(R5)xe2x80x94(CH2)nxe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4, xe2x80x94N(R5)xe2x80x94C(xe2x95x90O)xe2x80x94(CH2)nxe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4, xe2x80x94Oxe2x80x94(CH2)nxe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4, xe2x80x94N(R5)xe2x80x94(CH2)nxe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4, xe2x80x94Sxe2x80x94(CH2)nxe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4, xe2x80x94(CH2)m+1xe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4, xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94(CH2)nxe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4, and xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94(CH2)mxe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4 wherein n and m independently are integers from 0 to 8; where R3 is selected from the group consisting of xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94C(xe2x95x90S)xe2x80x94, xe2x80x94C(xe2x95x90S)xe2x80x94Oxe2x80x94, xe2x80x94N(R5)xe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94C(xe2x95x90O)xe2x80x94N(R5)xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94N(R5)xe2x80x94, optionally substituted C1-4 alkylene, optionally substituted arylene, optionally substituted heteroarylene, and optionally substituted C3-C7 cycloalkylene.
In such embodiments, R4 is the terminus of the linking unit and is a functional group suitable for chemo-, photo-, electro- or thermo-reactivity and combinations thereof. R4 as such, can be selected from the group comprising of activated forms of a carboxylates, carboxylic acid, amine, acyl halides, activated esters, thiols, disulphides (xe2x80x94Sxe2x80x94Sxe2x80x94R), halides, sulphonates, quinones and imides. However, as is known by the person skilled in the art, the covalent linking of two components can be accomplished by countless methods too numerous to describe or enumerate (see Hermanson, Mallia and Smith, Immobilized Affinity Ligand Techniques, Academic Press, 1992, San Diego, Calif., USA).
The coupling of a hapten to a polymer carrier can proceed via a multitude of methods, as is known by the person skilled in the art. The method will of course be dependent on the functional groups present on the polymer carrier. Thus, the nature of R4, and the nature of the terminus of R2 in general will depend on the desired coupling technique and on the choice of polymer carrier, and consequently on the functional groups present on the polymer carrier.
Ideally, however, the nature of R4, and moreover of R2 in general should be such that cross-reactivity with R1, X1, and X2 is avoided.
In other embodiments of the invention, the terminus of the linking unit is a functional group deemed suitable for activation for coupling to a polymer carrier. That is to say that the functional group must first be activated by chemical modification prior to being covalently linked to the polymer carrier. In such embodiments, the terminus of the linking unit is any functional group that can be readily activated by methods known by the person skilled in the art. Such functionalities can be selected from the group comprising of amines, carboxylic acids, thiol, hydroxyl and so forth. The person skilled in the art can easily envisage a multitude of functional groups that are suitable for activation, means of activation, as well as means of covalently linking the activated system to a polymer carrier.
In embodiments where the linking unit must first be activated by chemical modification prior to covalent linkage to the polymer carrier, R4, the terminus of the linking unit suitable for activation can be a function group selected from the group comprising of amines, carboxylic acids, thiol, hydroxyl and any functional group that can be readily activated by methods known by the person skilled in the art.
In certain embodiments, a covalent bond between the linker unit and an amine present on a polymer carrier, such as a protein, the terminus of the linker unit is a carboxylate or a carboxylic acid. In a preferred embodiment of the invention, the terminus of the linker unit of the hapten, such as R4, comprises of a carboxylic acid, which can be readily activated for coupling for immunisation purposes. Activation can be done by modifying the carboxylic acid to an activated form of a carboxyl such as, but not exclusively, to its corresponding acyl halide, anhydride, activated esters such as succinimidyl esters. The coupling of the linking unit may also be to groups other than amines present on a polymer carrier such as thiols and hydroxyls.
In preferred embodiments if this assay, the linking unit moiety is not pre-activated for coupling to a polymer carrier. In such systems, the terminus of the linker unit, such as R4, is a carboxylic acid, an amine, a thiol, or a hydroxyl. In embodiments wherein the polymer carrier is a protein or polyamide, the terminus of the linker unit, such as R4, is preferably a carboxylic acid, an amine, a thiol, or a hydroxyl, particularly an amine or carboxylic acid.
In another embodiment of this aspect of the invention, the terminus of the linking unit R4 is selected from a group comprising of amine, hydroxyl and thiol. These groups can couple to a carboxylate, carboxylic acid, or any form of a carboxyl, including activated forms, present on the polymer carrier. Preferred embodiments of this aspect of the invention, an amine functionality at the terminus of the linking unit is used to couple to a carboxylate, carboxylic acid, or any form of a carboxyl, including activated forms, present on the polymer carrier.
Prior to immunisation, the haptens must be conjugated to a polymer carrier, such as to a protein, for e.g. ovalbumin (OA). As stated above, in certain embodiments, conjugation may require preliminary activation of the terminal carboxyl of the linking unit (i.e. in non pre-activated systems). As is known by the person skilled in the art, activation of the carboxyl can be done using common peptide coupling reagents (see for e.g. M. Bodansky and A. Bodansky, The Practice of Peptide Synthesis, 2. Ed, Springer-Verlag, 1994, J. Jones,, xe2x80x9cThe Chemical Synthesis of Peptidesxe2x80x9d, Clarendon Press, 1991).
As described in Example 3, non pre-activated haptens in which the terminus of R2, i.e. R4, was a carboxylic acid were activated for coupling using reagents such as BOP and DCC and the appropriate additives and reagents. The activated hapten was reacted to a polymer carrier to form a hapten-polymer carrier complex prior to immunisation.
As is known by the person skilled in the art, the number of haptens bound to each polymer carrier can vary dramatically according to the conditions employed in the coupling process. Also, the type of polymer carrier used can influence the number of haptens bound to each polymer carrier. In preferred embodiments, the number of haptens per polymer carrier range from 1:1 to 50:1, such as 1:1 to 5:1, 2:1 to 10:1, 5:1 to 20:1, 10:1 to 30:1, 20:1 to 40:1, or 30:1 to 50:1. In one particular embodiment, the hapten per polymer carrier ratio was 5:1 with respect the number of moles of the hapten N-(5-carboxypentyl)-2,4-dichloro-3-amido aniline (EQ-0031) bound to the protein ovalbumin.
ii) The length and position of linking unit moiety
The present investigators have found that the position of the linking unit is critical to the ability of the hapten to raise antibodies suitable for the analysis of BAM (vide infra).
Specifically, the position of R2 relative to R1 (and in turn to X1 and X2) may be critical to appropriately orient the hapten for recognition by an antibody during immunisation. The inventors have critically determined that compounds where R2 (as opposed to R1) serves as the linking unit moiety are suitable to raise antibodies for the analysis of dichlorbenil and chlorthiamid degradation products. Thus R2 is in the meta position relative to R1.
As stated, the linking unit moiety must be in the meta position relative to R1. The importance of the position of the linking unit was illustrated in cases where immunisations with haptens in which R1 itself served as a linking unit (for instance, where the amide group of BAM was chemically extended in such as way as to yield succinimidyl-N-(2,6-dichlorobenzoyl)-6-amino-hexanoate, a pre-activated hapten (EQ-0025)). In such a case, the hapten was able to induce strong immunoresponse and several clones were generated. However, and most notably, none of the produced antibodies were able to detect free dichlorbenil nor its main degradation product, namely BAM, the preferred test analyte. This result highlights the importance of the position of the linking unit. This phenomenon clearly indicates that the antibodies have linking unit-specificity with regards to the linking unit""s position on the ring. This also implies that the antibodies may be recognising the linking unit (or at least a part thereof) as well as the ring structure. Thus neither X1 and X2 were used to link to the polymeric carrier.
The linker unit can consist of a variety of chains, including, but not limited to esters, amides, ethers, thioethers, and secondary amines. The linking unit moiety, R2 is of a length appropriate to suitable orient the hapten relative to the polymer carrier to create a recognition motif for an antibody.
Currently believed to be of critical importance for appropriate orientation of the hapten molecule are the flexibility and length of the linking unit component, R2. The linking unit serves to orient the hapten for recognition by the antibody. The linking unit, R2, in formula I, is preferably in the range of 3 to 25 atoms in length, excluding the atoms of R4, particularly ranging form 3-15 atoms in length. Alternatively, the length can be viewed in absolute empirical terms, namely as being between 5 and 75 xc3x85, preferably no more than 50 xc3x85.
Present understanding of such systems is that for appropriate orientation of the hapten molecule, the flexibility and length of the linking unit component, R2 are extremely pertinent. Given that R2 can be selected from the group consisting xe2x80x94C(xe2x95x90O)xe2x80x94N(R5)xe2x80x94(CH2)nxe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4, xe2x80x94N(R5)xe2x80x94C(xe2x95x90O)xe2x80x94(CH2)nxe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4, xe2x80x94Oxe2x80x94(CH2)nxe2x80x94R3, xe2x80x94(CH2)mxe2x80x94R4, xe2x80x94N(R5)xe2x80x94(CH2)nxe2x80x94R3xe2x80x94(CH2)mOxe2x80x94R4, xe2x80x94Sxe2x80x94(CH2)nxe2x80x94R3xe2x80x94(CH2)mR3xe2x80x94(CH2)mxe2x80x94R4, xe2x80x94(CH2)n+1xe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4, xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94(CH2)nxe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4 and xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94(CH2)nxe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4, the flexibility of the linking unit is regulated to a great extent by the nature of R3 and by the size of the terms m and n. In one embodiment, n and m are independent and are integers from 0 to 8. The greater the independent values and the greater their added values, the greater the length and flexibility of the linking unit. Their independent values can, in one embodiment, be as low as 0 resulting in a short linking unit moiety. Their added values (m+n), can range from 0 to 16, preferably being between 0 and 12, such as 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, and 0. Linking units having a total number of atoms between the aromatic moiety (formula I), and R4 in the range of 3-25 (not inclusively), such as in the range of 3-15, are believed to be particularly interesting.
In one embodiment of the invention, R2 is linked to the aromatic portion of hapten used for immunisation by an amide bond. The linkage can be either as the C-terminus or N-terminus of an amide bond (ie the carbon of the carbonyl portion of the amide bond is bound to the aromatic ring or the amine portion of the amide bond is bound to the aromatic ring of the hapten used for immunisation). Haptens from both embodiments have generated immune response in live animals. In the preferred embodiments the amine of the amine bond is bound to the ring.
The nature of R3 may also be of critical importance for appropriate orientation of the hapten molecule influencing the flexibility and length of the linking unit component. R3 is selected from the group consisting of xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94C(xe2x95x90S)xe2x80x94, xe2x80x94C(xe2x95x90S)xe2x80x94Cxe2x80x94, N(R5)xe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94C(xe2x95x90O)xe2x80x94N(R5)xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94N(R6)xe2x80x94, optionally substituted C1-4 alkylene, optionally substituted arylene, optionally substituted heteroarylene, an optionally substituted C3-C7 cycloalkylene.
Some functionalities, such as when R3 is a ring system (be they optionally substituted arylene, optionally substituted heteroarylene, an optionally substituted C3-C7 cycloalkylene) instill rigidity to the system. Other functionalities, such as when R3 is selected from xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, C(xe2x95x90O)xe2x80x94, xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94C(xe2x95x90S)xe2x80x94, xe2x80x94C(xe2x95x90S)xe2x80x94Oxe2x80x94, xe2x80x94N(R6)xe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94C(xe2x95x90O)xe2x80x94N(R5)xe2x80x94 cause variations in both the flexibility and orientation by causing a bend in a linking unit.
In preferred embodiments of the invention, R3 is selected from the group consisting of optionally substituted C1-4 alkylene, optionally substituted C3-7 cycloalkylene, optionally substituted aromatic and optionally substituted heteroarylene, in particular selected from optionally substituted C1-4 alkylene and optionally substituted C3-7 cycloalkylene, most particularly is C1-4 alkylene.
In the embodiment whereby R3 is selected from xe2x80x94N(R5)xe2x80x94, R5 is selected from hydrogen, optionally substituted C1-4 alkyl, optionally substituted C2-5 alkenyl, optionally substituted C2-8 alkynyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted C3-C7 cycloalkyl. In preferred embodiments, R5 consists of hydrogen, optionally substituted C1-4 alkyl, optionally substituted C2-5 alkenyl and optionally substituted C2-8 alkynyl, preferably from hydrogen and optionally substituted C1-4 alkyl.
iii The Polymer Carrier
As previously stated, prior to immunisation the hapten is coupled to a polymer carrier. As in known by the person skilled in the art, any number of polymer carriers are suitable for generating an immunoresponse in the immunisation procedure (see Christopher P. Price and David J. Newman eds., Principles and Practice of Immunoassay, second edition, p. 35-64, Macmillan Reference Ltd., 1997, London, U.K.). The polymer carrier can be selected from any carrier which elicits an immune response including but not limited to polyamides, proteins, nucleic acids, polynuclueotides, glycans, dextrans, peptido-glycans, lectins, glutins. The person skilled in the art is aware that the immunisation process can be accomplished by innumerable means using a hapten and a polymer carrier of sorts. In some preferred embodiments, the polymer carrier is a protein. Proteins may typically be selected from a group comprising of Ovalbumin (OA), Bovine Serum Albumin (BSA); Keyhole Limpet Haemocyanin (KLH), Purified Protein Derivative (PPD, derived from Mycobacterium tuberculosis), thyroglobulin, albumin, streptavidin and avidin, notably OA and PPD, BSA, and KLH.
b) The Aromatic Moiety
The present investigators have found that R1 comprises at least part of one of the recognition sites, i.e. is part of the recognition motif, by the antibody in the immunisation process. R1 is selected from group comprising of xe2x80x94C(xe2x95x90O)xe2x80x94N(R5)(R5), xe2x80x94N(R5)xe2x80x94C(xe2x95x90O)xe2x80x94OR5, xe2x80x94C(xe2x95x90O)xe2x80x94OR5, xe2x80x94N(R5)xe2x80x94C(xe2x95x90O)(R5), xe2x80x94N(R5)xe2x80x94C(xe2x95x90S)(R5), xe2x80x94C(xe2x95x90S)xe2x80x94N(R5)(R5), xe2x80x94N(R5)xe2x80x94C(xe2x95x90S)xe2x80x94N(R5)R5), xe2x80x94N(R5)xe2x80x94C(xe2x95x90O)xe2x80x94N(R5)(R5), xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94R5, xe2x80x94C(xe2x95x90S)xe2x80x94OR5, xe2x80x94Oxe2x80x94C(xe2x95x90S)xe2x80x94R5, xe2x80x94Oxe2x80x94R5, xe2x80x94N(R5)(R5), xe2x80x94Sxe2x80x94R5, xe2x80x94CH2xe2x80x94R5, xe2x80x94CN, and halogen; wherein R5 is independently selected from the group consisting of hydrogen, optionally substituted C1-4 alkyl, optionally substituted C2-5 alkenyl, optionally substituted C2-8 alkynyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted C3-C7 cycloalkyl, such as xe2x80x94C(xe2x95x90O)xe2x80x94N(R5)(R5) xe2x80x94N(R5)xe2x80x94C(xe2x95x90O)xe2x80x94OR5, xe2x80x94C(xe2x95x90O)xe2x80x94OR5, xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94R5, xe2x80x94C(xe2x95x90S)xe2x80x94OR5, xe2x80x94Oxe2x80x94C(xe2x95x90S)xe2x80x94R5, xe2x80x94Oxe2x80x94R5, xe2x80x94N(R5)(R5), xe2x80x94Sxe2x80x94R5, xe2x80x94CH2xe2x80x94R5, xe2x80x94CN, and halogen. Preferred embodiments of the invention are those where R1 is xe2x80x94C(xe2x95x90O)xe2x80x94N(R5)(R5), xe2x80x94C(xe2x95x90S)xe2x80x94N(R5)(R), xe2x80x94N(R5)xe2x80x94C(xe2x95x90O)xe2x80x94OR5 and xe2x80x94CN, preferably xe2x80x94C(xe2x95x90O)xe2x80x94N(R5)(R5), xe2x80x94C(xe2x95x90S)xe2x80x94N(R5)(R5) and CN, in particular xe2x80x94C(xe2x95x90O)xe2x80x94NH2 (i.e. where both R5 groups are hydrogen).
R1is situated between X1 and X2. In one embodiment of the invention, X1 and X2 are as present in one of the principal test analytes, BAM; i.e. both X1 and X2 are chlorine. However, X1 and X2 need not be identical. In another embodiment of the invention, X1 is chlorine and X2 is selected from bromine, fluorine and iodine; alternatively, X2 is chlorine and X1 is selected from bromine, fluorine and iodine. In an interesting embodiment of the invention, both X1 and X2 are independently selected from halogens other than those present in one of the principal test analyte (BAM), such as bromine, fluroine or iodine.
In a combination of preferred embodiments, X1 is chlorine, X2 is chlorine, and R1 is C(xe2x95x90O)xe2x80x94NH2.
One means by which the importance of the nature of R1 can be illustrated is by the comparison of the suitability of two haptens that differ solely in the nature of R1. When the hapten N-(5-Carboxypentyl)-2,4-dichloro-3-amido aniline (EQ-0031) bound to the protein ovalbumin was used for immunisation purposes, good responses were generated and several clones were produced. One of these clones (HYB 273-1) has been found to be suitable to generate monoclonal antibodies for the use of an immunoassay for BAM. Immunisations with a hapten (EQ-0030) in which the linking unit R2, is in the same position (meta) to R1 and of the same length as that used in EQ-0031, but where the ring substituents (X1, X2, and R1) are modified only in that R1 is a nitrile (CN) instead of an amide {xe2x80x94C(xe2x95x90O)xe2x80x94NH2)} were not able to induce appropriate immunoresponses. No suitable antibodies were generated. This result indicates that the judicious choice of X1, X2 and R1 are crucial for generating antibodies appropriate for the immunoassay of dichlorbenil and chlorthiamid and their derivatives and degradation products.
In an exemplary embodiment of the method according to the present invention monoclonal antibodies generated from the clone HYB 273-1, in turn generated from immunisations with hapten EQ-0031 were, as stated above, used in homologous competitive immunoassays for BAM. These antibodies demonstrated excellent selectivity showing very little cross-reactivity (less than 10.8%) with other analytes of similar structural composition (see Table 3). The cross-reactivity is determined by dividing the IC50 (vide infra) of the desired analyte (in this case BAM) by the IC50 of the test analyte and multiplying the total by 100.
The above example is not intended to be limiting in any way. Any complex according to the present invention may be used to raise antibodies. Antibodies raised from a complex according to the present invention may be used in the present invention
The cross reactivity of an assay is a measure of its specificity for the analyte relative to similar or related compounds. It is useful in assessing its accuracy in crude samples with several potential haptens. Standard curves based on the 4-parameter-logistic equation (eq 1) were generated for a series of analytes (Table 3) in the range 100 to 0 mg/L (10-fold titrations). The IC50 value (C*) for each analyte was then compared with the IC50 of BAM (C) in equation 3.                     CR        =                              (                          C                              C                *                                      )                    xc3x97          100          ⁢          %                                    (        3        )            
As can be seen by Table 3, the assay in which HYB 273-1 was used as a antibody, showed very high specificity for BAM. The highest cross-reactivity was with 2,4-dichlorobenzamide at 3.8%. There was no measurable cross-reactivity with the parent compound, dichlorbenil.
In preferred embodiments, the complex of N-(5-carboxypentyl)-2,4-dichloro-3-amido aniline and a protein serves to raise antibodies. The use of antibodies raised from the N-(5-carboxypentyl)-2,4-dichloro-3-amido aniline-protein is demonstrated in the Examples, such as antibodies cloned from HYB-273-1. However, as is known by the person skilled in the art, any antibody raised from a complex according to the present invention may be suitable for an assay according to the present invention.
As was discussed earlier, when R1 of the hapten exists as an amide which was homologated into a linking unit (succinimidyl-N-(2,6-dichlorobenzoyl)-6-amino-hexanoate, hapten EQ-0025), assay results were poor. Quite surprisingly however, immunisations with haptens in which the same linking unit as that used in EQ-0025 is in the meta-position with respect to the amide of BAM (such as EQ-0031), generated good immunoresponse and clones produced were used to generate monoclonal antibodies (as stated above) by the methods known by a person skilled in the art. Some of these cloned antibodies (such as HYB 273-1) have excellent specificity for the analyte BAM and are useful for immunoassays of type embodied in this invention. These results, on their own and when compared to results in which no immunoresponse was produced, indicate that the position and/or size of the linking unit are crucial for generating antibodies applicable to the immunoassay of dichlorbenil and chlorthiamid degradation products.
Live mice were primed for immunisation by an intraperitoneal injection of live BCG-vaccine one month before immunisation. The antigen (hapten-OA complex), once adsorbed onto Al(OH)3, was injected intraperitonally and test bleeding were collected. When antibody titre""s exceeded 1:2000, mice were considered ready for fusion and the method described by Kxc3x6hler and Milstein (1975) was employed with minor modifications. Monoclonal antibodies were selected on basis of titre, affinity and specificity. Selected clones were recloned until monoclonality and stored frozen at xe2x88x9280xc2x0 C.
II. The Compound Used for Immobilisation
i) The Hapten and Assay Types
In interesting embodiments of the method according to the present invention, the assays are adapted for high throughput screening. The assays may comprise but are not limited to assays utilising radioligand binding, using scintillation proximity assays; and fluorescence technologies such as time-resolved fluorescence, fluorescence resonance energy transfer, and fluorescence polarisation.
The immunoassay utilised in the identification or quantification of any of the degradation products of dichlorbenil and chlorthiamid may be, for example, a homogeneous immunoassay or a heterogeneous immunoassay procedure (see Christopher P. Price and David J. Newman eds., Principles and Practice of Immunoassay, second edition, p. 35-64, Macmillan Reference Ltd., 1997, London, U.K.).
In a heterogeneous immunoassay, an immobilised hapten is attached to an insoluble matrix. A heterogeneous assay involves the separation of the unbound antigen in a liquid phase from the bound hapten (on the solid phase). In a preferred embodiment of the invention, the assay is heterogeneous. Furthermore, in this preferred embodiment of the invention, the hapten used to raise antibodies in the animal is also the hapten used for immobilisation onto the solid support. This is known as a homologous (heterogeneous) competitive assay, as is known by the person skilled in the art.
In an alternative embodiment of a heterogeneous immunoassay, the antibody is immobilised to the solid support.
In embodiments wherein the immunoassay used for the identification or quantification of the degradation products of dichlorbenil and chlorthiamid is homogeneous, a solid phase support is not utilised but rather the assay completely solution based. In homogenous assays, all reagents are in the same phase and the reaction is not stopped and assay parameters are measured when the assay system has reached a steady state. They do not require a phase separation whereby the xe2x80x9cfreexe2x80x9d reagents are washed from those xe2x80x9cboundxe2x80x9d. In such an assay, the hapten is labelled with an enzyme and to this is added sample containing an unknown concentration of the analyte. An antibody specific to the hapten (and analyte if they are different, i.e. a heterologous homogeneous assay) is then added and the antibody binds to the enzyme-labelled hapten and to the analyte. The interaction between the antibody and the hapten results in the inhibition of the enzymatic activity of the label. A suitable substrate for the enzyme label is added and spectrophotometric analysis of the samples, once compared to the response of known concentrations of the analyte standards, produces a quantifiable result.
In embodiments wherein the hapten is immobilised to the solid support, the linking unit, R2, serves to couple the hapten to the solid support. This linking unit can be a pre-activated system or activated as described for the coupling of the hapten to a polymer carrier in the immunisation process. The hapten used for immunisation can itself be bound to the solid support. Conversely, this hapten can be immobilised to the solid support via a polymer carrier. In the latter case, the activated or pre-activated hapten is first coupled to the polymer carrier to form a hapten-polymer complex. This complex is immobilised to the solid support matrix for the purpose of the assay.
One embodiment of the method entails what a person skilled in the art knows as a sandwich assay. In this case, an antibody raised from the hapten-polymer carrier complex is immobilised on the solid phase. A sample containing the analyte is added an allowed to bind to the antibody. After appropriate incubation time and washing, a suitably labelled antibody is added to the solid phase and allowed to incubate and specifically bind to the analyte such that the analyte is sandwiched between two antibodies specific for it. All unbound labelled antibody is washed away. Enzymatic substrate is then added and colorimetric analysis allows for comparison to a standard procedure (see Christopher P. Price and David J. Newman eds., Principles and Practice of Immunoassay, second edition, p. 35-64, Macmillan Reference Ltd., 1997, London, U.K.).
In a preferred embodiment of the invention, the hapten used for immobilisation is modified (relative to the hapten used for immunisation) such that the affinity of an antibody is shifted from the immobilised hapten to the analyte, which resembles the hapten utilised for immunisation. Such an immunoassay is called a heterologous competitive assay and is more sensitive than the homologous competitive immunoassay.
Of course, in scenarios where the hapten used for immunisation is different to that used for immobilisation (a heterologous assay), the antibodies raised from particular hapten must be able to recognise the immobilised hapten as well as the test analyte. An immobilised hapten of the general formula II allows for greater affinity for the intended analytes (i.e. degradation products of dichlorbenil and chlorthiamid) by antibodies raised from haptens of the general formula I. The immobilised hapten can differ in that it comprises of a heteroaromatic ring, in the judicious choice of substituents around the ring, and in the choice of linking unit. Alteration of the ring system and ring periphery allows for attenuation as to the sensitivity of the assay.
As it has been discussed, the hapten used for immobilisation need not necessarily be identical to the hapten used for immunisation (heterologous assay). An assay in which the affinity of the antibody is higher for the analyte than for the immobilised hapten is more sensitive than its homologous counterpart. It allows for an exploitation of a more diverse range of haptens utilised for immobilisation. One can conceive of immobilised haptens consisting of, for instance, substitution not only of the groups attached to the ring, i.e. X1, X2, R1, R2 of formula I, but also to the ring itself, insofar as that the immobilised hapten comprises of a heteroaromatic rings, to comprise of haptens of the general formula II. 
In this embodiment, a method for an immunoassay has a hapten of the general formula II immobilised to a solid support; wherein Z1 is selected from Cxe2x80x94R8 or nitrogen, Z2 is selected from Cxe2x80x94R7 or nitrogen, Z3 is selected from Cxe2x80x94R8 or nitrogen, Z4 is selected from Cxe2x80x94R9 or nitrogen, where at least one of R6-R10 is present and serves as a linking unit for immobilisation of the hapten to the solid support, and the ones of R6-R10 which are present and do not serve as a linking unit are independently selected from a group consisting of xe2x80x94C(xe2x95x90O)xe2x80x94NH2, xe2x80x94N(R5)(R5), xe2x80x94N(R5)xe2x80x94C(xe2x95x90O)(R5), xe2x80x94N(R5)xe2x80x94C(xe2x95x90S)(R5), xe2x80x94C(xe2x95x90S)xe2x80x94N(R5)(R5), xe2x80x94N(R5)xe2x80x94C(xe2x95x90S)xe2x80x94N(R5)(R5), xe2x80x94N(R5)xe2x80x94C(xe2x95x90O)xe2x80x94N(R5)(R5), xe2x80x94CN, xe2x80x94C(xe2x95x90O)xe2x80x94N(R5)(R6), xe2x80x94N(R5)xe2x80x94C(xe2x95x90O)xe2x80x94OR5, xe2x80x94N(R5)xe2x80x94C(xe2x95x90O)xe2x80x94OR5, xe2x80x94C(xe2x95x90O)xe2x80x94OR5, xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94R5, xe2x80x94C(xe2x95x90S)xe2x80x94OR5, xe2x80x94Oxe2x80x94C(xe2x95x90S)xe2x80x94R5, hydrogen, and halogen; where X1 is selected from halogens, and where R5 is independently selected from the group consisting of hydrogen, optionally substituted C1-4 alkyl, optionally substituted C2-5 alkenyl, optionally substituted C2-6 alkynyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted C3-C7 cycloalkyl.
The compound used for immobilisation can comprise of a heteroaromatic moiety, according to general formula II, which can comprise of up to 4 heteroatoms in the ring. In one embodiment, the number of heteroatoms is less than 4, such as less than 3, such as 2, or preferably 1 or 0. The position of the heteroatom relative to the fixed position of the carbon bound to X1 may play an important role in the ability of the immobilised hapten to be recognised by the antibody raised from a hapten of the general formula I. In a another embodiment, no two neighbouring atoms are heteroatoms. In another preferred embodiment Z1-Z4 respectively consist of Cxe2x80x94R8 to Cxe2x80x94R9, that is to say, the ring is not heteroaromatic.
The groups R8-R10 serving as linking units can be independently selected from the group consisting of xe2x80x94C(xe2x95x90O)xe2x80x94N(R5)xe2x80x94(CH2)nxe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4, xe2x80x94N(R5)xe2x80x94C(xe2x95x90O)xe2x80x94(CH2)nxe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4, xe2x80x94Oxe2x80x94(CH2)nxe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4, xe2x80x94N(R5)xe2x80x94(CH2)nxe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4, xe2x80x94Sxe2x80x94(CH2)nxe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4, xe2x80x94(CH2)n+1xe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4, xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94(CH2)nxe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4, and xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94(CH2)nxe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4 wherein n and m independently are integers from 0 to 8; where R3 is selected from the group consisting of xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94C(xe2x95x90S)xe2x80x94, xe2x80x94C(xe2x95x90S)xe2x80x94Oxe2x80x94, xe2x80x94N(R5)xe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94C(xe2x95x90O)xe2x80x94N(R5)xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94N(R5)xe2x80x94, optionally substituted C1-4 alkylene, optionally substituted arylene, optionally substituted heteroarylene, an optionally substituted C3-C7 cycloalkylene; where R4 is selected from the group consisting of activated forms of a carboxyl, carboxylic acid, amine, acyl halides, activated esters, thiols, disulphides (xe2x80x94Sxe2x80x94Sxe2x80x94R), halides, sulphonates, quinones and imides; and where R5 is independently selected from the group consisting of hydrogen, optionally substituted C1-4 alkyl, optionally substituted C2-5 alkenyl, optionally substituted C2-6 alkynyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted C3-C7 cycloalkyl.
Given that no fewer than 1 of R6-R10 are selected from the group consisting of of xe2x80x94C(xe2x95x90O)xe2x80x94N(R5)xe2x80x94(CH2)nxe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4, xe2x80x94Oxe2x80x94(CH2)nxe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4, xe2x80x94N(R5)xe2x80x94(CH2)nxe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4, xe2x80x94N(R5)xe2x80x94C(xe2x95x90O)xe2x80x94(CH2)nxe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4, xe2x80x94Sxe2x80x94(CH2)nxe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4, xe2x80x94CH2xe2x80x94(CH2)nxe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4, xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94(CH2)nxe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4, and xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94(CH2)nxe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4, the potential of linking units is as much as 4. In preferred embodiments, fewer than 3 of R6-R10 serve as linking units, particularly fewer than 2, namely preferably 1.
In preferred embodiments, one of R6-R10 is a linking unit selected from the group consisting of xe2x80x94C(xe2x95x90O)xe2x80x94NHxe2x80x94(CH2)nxe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4 and xe2x80x94NHxe2x80x94C(xe2x95x90O)xe2x80x94(CH2)nxe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4, particularly from xe2x80x94NHxe2x80x94C(xe2x95x90O)xe2x80x94(CH2)nxe2x80x94R3xe2x80x94(CH2)mxe2x80x94R4.
The terminus of the linker unit R6-R10, such as R4, is a functional group suitable for chemo-, photo-, electro- or thermo-reactivity and combinations thereof. The terminus of the linker unit or R4 as such, can be selected from the group comprising of activated forms of a carboxylates, carboxylic acid, amine, acyl halides, activated esters, thiols, disulphides (xe2x80x94Sxe2x80x94Sxe2x80x94R), halides, sulphonates, quinones and imides. Alternatively, as discussed infra, the terminus of the linker unit or R4 as such can be a group suitable for activation such as a carboxylic acid, amine, thiol, or hydroxyl. However, as is known by the person skilled in the art and as discussed infra, the covalent linking of two components can be accomplished by countless methods too numerous to describe or enumerate.
Those of R6-R10 not serving as linking units are selected from a group comprising of xe2x80x94C(xe2x95x90O)xe2x80x94NH2, xe2x80x94N(R5)(R5), xe2x80x94N(R5)xe2x80x94C(xe2x95x90O)(R5), xe2x80x94N(R5)xe2x80x94C(xe2x95x90S)(R6), xe2x80x94C(xe2x95x90S)xe2x80x94N(R5)(R5), xe2x80x94N(R5)xe2x80x94C(xe2x95x90S)xe2x80x94N(R5)(R5), xe2x80x94N(R5)xe2x80x94C(xe2x95x90O)xe2x80x94N(R5)(R5), xe2x80x94CN, xe2x80x94N(R5)xe2x80x94C(xe2x95x90O)xe2x80x94OR5, xe2x80x94C(xe2x95x90O)N(R5)(R5), xe2x80x94C(xe2x95x90O)xe2x80x94OR5, xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94R5, xe2x80x94C(xe2x95x90S)xe2x80x94OR5, xe2x80x94Oxe2x80x94C(xe2x95x90S)xe2x80x94R5, hydrogen and halogen. In one embodiment, at least two of R6-R10 not serving as linking units are selected from a group comprising xe2x80x94C(xe2x95x90O)xe2x80x94NH2, xe2x80x94N(R5)(R5), xe2x80x94C(xe2x95x90O)xe2x80x94N(R5)(R5), xe2x80x94(R5)xe2x80x94C(xe2x95x90O)xe2x80x94OR5, xe2x80x94C(xe2x95x90O)xe2x80x94OR5, xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94R5, xe2x80x94C(xe2x95x90S)xe2x80x94OR5, xe2x80x94Oxe2x80x94C(xe2x95x90S)xe2x80x94R5, xe2x80x94N(R5)xe2x80x94C(xe2x95x90O)(R5), xe2x80x94N(R5)xe2x80x94C(xe2x95x90S)(R5), xe2x80x94C(xe2x95x90S)xe2x80x94N(R5)(R5), xe2x80x94N(R5)xe2x80x94C(xe2x95x90S)xe2x80x94N(R5)(R5), xe2x80x94N(R5)xe2x80x94C(xe2x95x90O)xe2x80x94N(R5)(R5), xe2x80x94CN and halogen. Within this embodiment, those R6-R10 not serving as linking units are preferably selected from xe2x80x94C(xe2x95x90OH)xe2x80x94NH2, xe2x80x94C(xe2x95x90O)xe2x80x94N(R5)(R5), xe2x80x94N(R5)xe2x80x94C(xe2x95x90O)xe2x80x94OR5, xe2x80x94N(R5)xe2x80x94C(xe2x95x90O)(R5), xe2x80x94N(R5)xe2x80x94C(xe2x95x90O)xe2x80x94N(R5)(R5), xe2x80x94CN, and halogen.
The number of halogens attached to the ring can be at most 4 (given that X1 is a halogen and at least one must be a linking unit). In a preferred embodiments of the invention, the number of halogens bound to the ring of the hapten used for immobilisation is at most 3, preferably 2 or 1, preferably 2.
The number of hydrogens bound to the ring can be at most 4, preferably no more than 3, most preferably 2 or 1, preferably 2.
The preferred embodiments of R3, R4, R5 and X1 described for compounds used for the raising of antibodies also apply to compounds used for immobilisation. Thus, in a preferred embodiment, the compound used for immobilisation is for the general formula I.
Moreover, not only can the hapten used for immobilisation be different than that used for immunisation, but the polymer carrier to which the respective haptens are bound to form a hapten-polymer carrier complex may be different. They may be different in type, or of the same type but of different structure, as for e.g. one can be a protein, polyamide or polymer whilst the other is a glycan of sorts or they can both be proteins but one being OA and the other streptavidin or PBD or biotin. The polymeric carrier used in the immobilisation process may be the same as that used for immunisations but in preferred embodiments they are different.
Polymer carriers suitable for the immobilisation procedure can be selected from those deemed suitable for the immunisation procedure.
ii) Solid Support and Immobilisation
In a heterogeneous immunoassay, the hapten is linked either directly or indirectly (vide infra) to a solid support. The solid support can be agarose, cellulose, silicone-rubber, glass, plastic in many configurations such as polystyrene beads and polystyrene microtitre plates and any such support known to the person skilled in the art procedure (see Christopher P. Price and David J. Newman eds., Principles and Practice of Immunoassay, second edition, Macmillan Reference Ltd., 1997, London, U.K.). In preferred embodiments of the invention, the heterogeneous immunoassay involves the use of polystyrene beads or polystyrene microtitre plates, in particular polystyrene microtitre plates, as solid support.
The solid surface to which the hapten is immobilised can be selected from a wide variety of solid surfaces used in the analytical and diagnostic fields. Some more interesting types of solid surfaces are those of organic polymers, glasses, silicium and silicium oxide (silica) as well as composite materials thereof.
Among the organic polymers, polystyrene, polycarbonate, polypropylene, polyethylene, cellulose, nitrocellulose, agarose, polyethyleneglycol terephthalate, polyvinylacetate, polyvinyldifluoride, polymethylpentene, polyvinylpyrrolidinone, polyacrylate, polyacrylonitrile, polymethylmethacrylate and polyvinylchloride are illustrative examples, where polystyrene and polycarbonate are especially interesting examples.
Among the glasses and ceramics, borosilicate glass (Pyrex glass) and soda-lime glass are especially relevant examples, e.g. in the form of specimen tubes, vials, and slides for microscopy.
The body of the solid surface in itself may have a form or may be designed and shaped for the particular desired use; e.g. the body may be in the form of a sheet, a film, a bead, a pellet, a disc, a plate, a ring, a rod, a net, a membrane, a filter, a tray, a microplate (a microtitre plate), a stick, or a multi-bladed stick. Especially interesting bodies to be coated according to the present invention are microplates (microtitre plates), e.g. polystyrene microplates (microtitre plates), sticks, slides, tubes and beads.
Immobilisation of the hapten to the solid support, either directly or via a polymer carrier, is accomplished by passive adsorption or covalent immobilisation by methods known to the person skilled in the art (Hermanson, 1992). In one embodiment of the immunoassay, the hapten is immobilised to a polystyrene microtitre plate by covalent immobilisation by means known to the person skilled in the art (see Example 1e). This immobilisation process is attractive in that it requires very little incubation time and was highly stable under dry storage (FIG. 4). In an alternative and particularly attractive embodiment of the method, the hapten is immobilised by passive adsorption. This adsorption process was also especially attractive in that immunoassays performed on these plates had an low coefficient of variation (CV), being less than 16% (FIG. 3). Preferred embodiments of the invention have CV values less than 20%.
As mentioned, the method of preparation of the microtitre plates varied depending of the method of immobilisation of the hapten. In a particularly attractive embodiment of covalent immobilisation (FIG. 2A), the immobilisation is performed through a photochemically active moiety, such as an anthraquinone moiety. This hapten, N-(5-carboxymethyl-pentyl)-2-anthraquinonecarboxamide (EQ-0028) was conjugated to a pre-existing hapten-protein complex (prepared in the usual manner between EQ-0031 and ovalbumin (OA)) to form hapten-OA-Anthraquinone conjugate complex. This complex was dialysed against PBS. Previous work by the applicant demonstrated that anthraquinones can be photocoupled to solid matrices by low energy photoactivation (PCT/DK 96/00167). Accordingly, a PolySorp plate was filled by a stationary dispensor with the hapten-OA-Anthraquinone conjugate. After exposing the plate to UV-light (300-400 nm) for 30 minutes, immobilisation was complete and the plate was washed and dried. The plate was either used immediately or sealed and stored at room temperature in the dark until use.
This latter method of immobilisation of complex, i.e. photocoupling via a conjugate complex comprising of anthraquinone, is a particular attractive embodiment. The anthraquinone moiety is directly coupled to the solid support and is linked through a polymer carrier to the hapten most directly accessible by an antibody. As was the case for haptens, the ratio of anthraquinone photocatalytic coupling moieties per polymer carrier molecule can range from 1:1 to 50:1. Preferred embodiments have said ratios no less than 2:1. In one particular embodiment where the anthraquinone photocatalytic coupler EQ-0028 was coupled to a pre-existing EQ-0031-OA complex (see Example 3), the ratio of EQ-0028 was 10 to 1 with respect to the number of moles of OA.
Passive adsorption (FIG. 2B) involved filling a MaxiSorp(trademark) plate with a stationary dispensor with the hapten-OA conjugate. After coating overnight at 4xc2x0 C., the plate was washed and dried. The plate was either used immediately or sealed and stored at room temperature in the dark until use.
As stated, the assays may be homogeneous or heterogenous. In embodiments wherein the immunoassay used for the identification or quantification of the degradation products of dichlorbenil and chlorthiamid is homogeneous, a solid phase support is not utilised but rather the assay completely solution based. In such an assay, the hapten is labelled with an enzyme and to this is added sample containing an unknown concentration of the analyte. An antibody specific to the hapten (and analyte if they are different, i.e. a heterologous homogeneous assay) is then added and the antibody binds to the enzyme-labelled hapten and to the analyte. The interaction between the antibody and the hapten results in the inhibition of the enzymatic activity of the label. A suitable substrate for the enzyme label is added and spectrophotometric analysis of the samples, once compared to the response of known concentrations of the analyte standards, produces a quantifiable result.
Standards were used to generate comparative standard curves. The standards were added to hapten-coated plates followed by a known amount of antibody, such as HYB 273-1 in buffer. The plate was incubated for 1 hour, washed, and incubated with Rxcex1MHRP, an enzyme label and incubated for 1 hour. After extensive washing, the enzyme substrate TMB-Plus was added. The colour development was stopped using acid and the absorbance read at 450 nm.
The standards were used to generate a semi-log graph based on the 4-parameter-logistic method (Rodbard, 1974: equation 1).                     Y        =                                            (                              A                -                D                            )                                      [                              1                +                                                      (                                          X                      C                                        )                                    B                                            ]                                +          D                                    (        1        )            
X: concentration of analyte (xcexcg/L).
Y: absorbance at 450 nm (A450).
A: maximum absorbance (upper asymptote).
D: minimum absorbance (lower asymptote).
C: midpoint between A and D. Extrapolation to x-axis yields the value of IC50 (xcexcg/L).
B: slope for the transition between A and D
The signals were normalised according to equation 2                               %          ⁢                      xe2x80x83                    ⁢                      B                          B              0                                      =                                            A              -                              A                xs                                                                    A                0                            -                              A                xs                                              xc3x97          100                                    (        2        )            
B: concentration of bound antibody and
B0: concentration of bound antibody in the absence of analyte.
On the plot, 100% corresponds to the absorption of the zero control (A0: 0 xcexcg/L) and 0% corresponds to the absorption of an excess control (Axa: 100 xcexcg/L). The normalised signals were plotted vs. BAM concentration and the four-parameter-logistic equation (eq 1) was fitted to the experimental points.
The standard curves were prepared by quadruple determinations in order to also establish the coefficient of variation. The coefficient of variation is a reflection of the reliability of an assay. They are determined by the mean reading at a particular analyte concentration over repeated assays over a range of concentrations. FIG. 3 shows the variations of normalised readings of quadruple determinations for the standard curves for the plates which were covalently immobilised as compared to the plates in which passive adsorption was the means of adsorbing hapten. The CVmean for covalently immobilised plates was 3.36% whereas that for passive adsorption plates was 15.20%. Preferred embodiments of this immunoassay have CVs less than 20%. The immunoassays described by this invention using haptens of the general formula I to generate antibodies were performed on plates which were antigen-coated by either passive adsorption or covalent immobilisation showed excellent detection limits. Detection limits for either method of immobilisation ranged from 0.0156 to 2 xcexcg/L. Detection limits are defined as DL15 which quantifies the analyte concentration giving a 15% decrease in maximum signal. Thus, the lower the numerical value of the detection limit of the test, the better the performance of the test. Considering the EU limit of 0.1 xcexcg/L for pesticides and their degradation products in drinking water, this assay is suitable for large-scale screening of samples of BAM. Preferred embodiments of the method according to the present invention have DL15 values of most 0.1 xcexcg/L, such as 0.1, 0.09, 0.08, or 0.06 xcexcg/L, preferably at most 0.05 xcexcg/L, such as 0.05, 0.04, 0.03, 0.02, or 0.01 xcexcg/L.
The midpoint between the maximum and minimum adsorbance, referred to as the IC50, indicates the concentration of analyte required causing a 50% signal reduction. It is a measure of the sensitivity of the assay. Again, the lower the numerical value of the IC50, the better the performance of the assay. For the covalently immobilised plates, the IC50 was found to be 0.189 xcexcg/L whereas for passively adsorbed plates, the IC50 was measured to be 0.144 xcexcg/L. Considering the EU-limit of 0.1 xcexcg/L for pesticides in drinking water, this immunoassay is suitable for large-scale screenings of water samples. Preferred embodiments of the invention have IC50 values at most 0.5 xcexcg/L, such as 0.5 or 0.4 xcexcg/L, preferably at most 0.3 xcexcg/L, such as 0.3, 0.2 xcexcg/L.
Samples collected from 3 different water types were spiked with BAM. The samples were spiked with 0.05, 0.1, 0.5 and 1 xcexcg/L of BAM and then analysed 4 times over 4 days (Table 2). The data showed that the assay was reliable over the investigative range (0.5 to 1 xcexcg/L) and had an average inter-assay coefficient variation of 4.3%.
Of notable importance is the absence of significant matrix effects for the two ground water types as compared to the Milli-Q(copyright) water. Thus the assay lends itself readily to a variety of water types.
Samples of unknown concentrations were assayed and their concentrations were determined by comparing normalised absorbency readings to those on the standard curve. Assay results of these unknown samples were compared to concentrations as ascertained by the standard techniques of BAM analysis, i.e. HPLC and GC-MS and a comparative analysis is shown in FIG. 5. This comparative analysis demonstrates the accuracy and reliability of the assay as described by this invention.
III. The Analyte
The method described by the invention is for the identification or quantification of degradation products of pesticides that, in themselves, or whose degradation products are recognised by antibodies raised by antibodies from haptens as described infra. More particularly, the method is for the identification or quantification of dichlorbenil and chlorthiamid and their degradation products. This herbicide is degraded in the hydrosoil by biotic or abiotic processes. In one embodiment of the invention, the method is limited to the identification or quantification of its major degradation products 2,6-dichlorobenzamide (BAM), 3-hydroxy-2,6-dichlorobenzamide and its sugar conjugates and 4-hydroxy-2,6-dichlorobenzamide and its sugar conjugates. In preferred embodiments, the assay is limited to the identification or quantification of 2,6-dichlorobenzamide (BAM), 3-hydroxy-2,6-dichlorobenzamide and its sugar conjugates. Alternatively, the method is limited to the identification or quantification of 2,6-dichlorobenzamide (BAM), 3-hydroxy-2,6-dichlorobenzamide and 4-hydroxy-2,6-dichlorobenzamide. Most preferably, the method is for the identification or quantification of 2,6-dichlorobenzamide (BAM).
IV. Synthesis of Haptens
Haptens were synthesised using methods known by the person skilled in the art (Example 2). The synthesis generally involved the coupling of the a carboxylic acid portion of a linking unit chain moiety to an amino function of a substituted benzene, or conversely, coupling an amine function of a linking unit chain moiety to a carboxylic acid functionality of a substituted benzene ring, using traditional peptide coupling procedures including the use of such reagents as DCC (dicyclohexylcarbodiimide, or other suitable carbodiimides), BOP (including BOP Reagent and BOP-CI) and the necessary additives in suitable solvents (see eg. M. Bodansky and A. Bodansky, xe2x80x9cThe Practice of Peptide Synthesisxe2x80x9d 2. Ed. Springer-Verlag, 1994, J. Jones, xe2x80x9cThe Chemical Synthesis of Peptidesxe2x80x9d, Clarendon Press, 1991).
V. Use of a Kit
In another aspect, the invention further relates to a kit for the immunological testing of pesticides, specifically to a kit for an immunoassay comprising a solid support and hapten as defined by formula II immobilised to the solid support. Moreover, the kit may further comprise an antibody raised from a hapten-polymer carrier complex as described by formula I.